Breast cancer is a deadly disease in modern society with limited therapeutics options. Tumor malignancy is likely due to tumor-initiating cells that are relatively undifferentiated. This project will develop an assay to identify chemical compounds that encourage differentiation of breast tumor cells, a strategy that has proven effective for other cancer types. The Microscopic Imaging of Epigenetic Landscape Breast Tumor Differentiation (MIEL-BTD) assay will utilize Vala Sciences? Structured Illumination Microscope (IC200-SIM) to quantify changes in the patterns of histone tags within the nucleus, associated with the initiation of differentiation of breast epithelium towards milk production. Phase I of this Fast-Track project will demonstrate feasibility utilizing MCF7 cells (representative of ER+ lumenal breast tumor), SKBR-3 cells (HER2/neu+) and MDA-MB-468 (triple negative) breast tumor cells, in standard, 2-D cultures using a version of the MIEL-BTD based upon immunolabeling. Phase II will develop genetically-encoded epigenetic probes (GEEPs) and tumor spheroid (3-D) culture models. SIM and image analysis methods will be developed for increased resolution of sub-nuclear patterns of epigenetic markers. The MIEL-BTD assay will be utilized in contract research for discovery of novel breast cancer therapeutics. The IC-200 SIM will be marketed to both academic and pharmaceutical cancer researchers.